We are working to establish an in vitro system for the study of checkpoint activation by the DNA damage response systems operating in human cells. As a model system, we are investigating checkpoint activation mediated by the DNA mismatch repair proteins MutSalpha and MutLalpha in response to several DNA damaging agents including SN1 alkylators, fluorouracil, and cisplatin. DNA damage sensors that target specific types of DNA damage activate the ATR and ATM protein kinases. This, in turn, activates a cascade of signaling kinases including Chk1 and Chk2 that ultimately result in cell cycle arrest at the G2/M boundary. We are purifying recombinant human proteins involved in this damage response with the goal of defining the underlying molecular mechanism. We are also interested in identifying components of key protein complexes that function to license cell cycle arrest in response to DNA damaging agents.